PROJECT SUMMARY Emotional behaviors, such as defensive responses to threat, are often dysregulated in mental illness. Currently, there is a poor understanding of the neural mechanisms that control transitions between modes and magnitudes of defensive responding. Obtaining such knowledge will provide significant progress toward the long-term goal of providing foundational knowledge used for the development of better therapeutics for mental illness. The overall objective of this application is to identify how distributed neuronal circuits control transitions to active forms of defensive behav- ior. The central hypothesis is that a widespread brain network that includes the central amygdala (CEA), ventromedial hypothalamus (VMH), periaqueductal grey (PAG), insular cortex (IC), and ventral hippocampus (vHIPP) controls defensive response transitions. The rationale that under- lies this proposal is that an enhanced understanding of these complex behavioral states and cir- cuits will provide foundational data for understanding multiple psychiatric disorders and their comorbidity. The proposed research tests the central hypothesis with three specific aims: 1) Iden- tify the functional role of specific projection pathways in defensive action selection; 2) Determine the impact of structures afferent to the CEA on flight behavior; and 3) Define the neuronal activity patterns that encode shifts in defensive states from freezing to flight to panic. The first aim will use viral vector delivery strategies and optogenetics to target and manipulate specific output path- ways of the CEA. The second aim will use retrograde targeting strategies to manipulate afferents to the CEA. Finally, the third aim will use deep-brain calcium imaging to monitor neural activity patterns in specific populations of projection neurons while mice transition through escalating lev- els of defensive responding. Successful completion of the proposed research will define mecha- nisms by which widespread brain networks mediate switching to active modes of defensive be- havior. The proposed research is innovative because it substantially departs from the status quo by investigating how the brain coordinates transitions between defensive behaviors. This will be significant because it will lay a foundation that facilitates understanding of the neuronal circuit basis of the maladaptive responses associated with mental illness. Indeed, this novel systems neuroscience approach to understanding how fear states are encoded will open new avenues of research into the neurobiological underpinnings of anxiety- and trauma-related mental health dis- orders, such as posttraumatic stress and panic disorder.